bioRxiv preprint doi: https://doi.org/10.1101/2020.10.23.350348; this version posted October 23, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Transferrin receptor is another receptor for SARS-CoV-2 entry Xiaopeng Tang1†, Mengli Yang2†, Zilei Duan1†, Zhiyi Liao1, 5†, Lei Liu4†, Ruomei Cheng1, 5, Mingqian Fang1, 5, Gan Wang1, 6, Hongqi Liu2, Jingwen Xu2, Peter M Kamau1, 6, Zhiye Zhang1, 6, Lian Yang3, Xudong Zhao4*, Xiaozhong Peng2*, Ren Lai1, 6, 7* 1Key Laboratory of Animal Models and Human Disease Mechanisms of Chinese Academy of Sciences/Key Laboratory of Bioactive Peptides of Yunnan Province, KIZ-CUHK Joint Laboratory of Bioresources and Molecular Research in Common Diseases, National Resource Center for Non-Human Primates, Kunming Primate Research Center, National Research Facility for Phenotypic & Genetic Analysis of Model Animals (Primate Facility), Sino-African Joint Research Center, and Center for Biosafety Mega-Science, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming 650107, Yunnan, China; 2Institute of Medical Biology, Chinese Academy of Medical Sciences, Peking Union Medical College, Kunming 650031, China; 3Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650204, China; 4laboratory of animal tumor models, Frontiers Science Center for Disease-related Molecular Network, State Key Laboratory of Biotherapy and Cancer Center, National Clinical Research Center for Geriatrics, West China Hospital, Sichuan University, Chengdu 610041, China; 5Kunming College of Life Science, University of Chinese Academy of Sciences, Kunming 650204, Yunnan, China; 1 bioRxiv preprint doi: https://doi.org/10.1101/2020.10.23.350348; this version posted October 23, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 6Institute for Drug Discovery and Development, Chinese Academy of Sciences, Shanghai 201203, China; 7Lead Contact. †These authors contributed equally to this work. *Corresponding authors: [email protected] (R.L.), [email protected] (X.P.) or [email protected]. 2 bioRxiv preprint doi: https://doi.org/10.1101/2020.10.23.350348; this version posted October 23, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Abstract Angiotensin-converting enzyme 2 (ACE2) has been suggested as a receptor for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) entry to cause coronavirus disease 2019 (COVID-19). However, no ACE2 inhibitors have shown definite beneficiaries for COVID-19 patients, applying the presence of another receptor for SARS-CoV-2 entry. Here we show that ACE2 knockout dose not completely block virus entry, while TfR directly interacts with virus Spike protein to mediate virus entry and SARS-CoV-2 can infect mice with over-expressed humanized transferrin receptor (TfR) and without humanized ACE2. TfR-virus co-localization is found both on the membranes and in the cytoplasma, suggesting SARS-CoV-2 transporting by TfR, the iron-transporting receptor shuttling between cell membranes and cytoplasma. Interfering TfR-Spike interaction blocks virus entry to exert significant anti-viral effects. Anti-TfR antibody (EC50 ∼16.6 nM) shows promising anti-viral effects in mouse model. Collectively, this report indicates that TfR is another receptor for SARS-CoV-2 entry and a promising anti-COVID-19 target. 3 bioRxiv preprint doi: https://doi.org/10.1101/2020.10.23.350348; this version posted October 23, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Introduction Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) which has been assessed and characterized as a pandemic by world health organization on 11 March, 2020 (https://www.who.int/), causes coronavirus disease-19 (COVID-19) with influenza-like manifestations ranging from mild disease to severe pneumonia, fatal acute lung injury, acute respiratory distress syndrome, multi-organ failure, and consequently resulting to high morbidity and mortality, especially in older patients with other co-morbidities1-10. As of October 22, 2020, the ongoing COVID-2019 pandemic has swept through 212 countries and infected more than 40 932 220 individuals, posing an enormous burden to public health and an unprecedented effects to civil societies. Unfortunately, to date, there is no vaccine or antiviral treatment for this coronavirus. The pathogenesis and etiology of COVID-19 remain unclear, and there are no targeted therapies for COVID-19 patients 11. Pioneer studies 12,13 have demonstrated that angiotensin converting enzyme 2 (ACE2) is the critical receptor for severe acute respiratory syndrome coronavirus (SARS-CoV), which first emerged 17 years ago 14. The spike protein of SARS-CoV binds to the host ACE2 receptor and then enters into the target cells. SARS-CoV-2 bears an 82% resemblance to the genomic sequence of SARS-CoV15. Especially, the receptor binding domain (RBD) of SARS-CoV-2 is highly similar to the SARS-CoV RBD, suggesting a possible common host cell receptor. Several cryoelectron microscopy (cryo-EM) studies have demonstrated that SARS-CoV-2 spike protein directly binds to ACE2 with high affinity 16-20. Soluble ACE2 fused to Ig 18 or a 4 bioRxiv preprint doi: https://doi.org/10.1101/2020.10.23.350348; this version posted October 23, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. nonspecific protease inhibitor (camostat mesylate) showed ability to inhibit infections with a pseudovirus bearing the S protein of SARS-CoV-2 21. Camostat mesylate of high doses (100 mg/mL) has been reported to partially reduce SARS-CoV-2 growth 21. Recently, Monteil et al., reported that clinical-grade soluble human ACE2 can significantly block early stages of SARS-CoV-2 infections in engineered human tissues 19. However, no definite evidence indicates that taking ACEIs/ARBs is beneficial or harmful for COVID-19 infected patients 11, suggesting possibilities of other factor/factors assisting in virus entry. Indeed, recent study has shown that neuropilin-1 facilitates SARS-CoV-2 cell entry and infectivity 22. Here, we identified the ubiquitously expressed transferrin receptor (TfR), which is co-localized with ACE2 on cell membranes, as an entry factor of SARS-CoV-2 by directly binding to virus spike protein and ACE2 with high affinities. Moreover, we found that interferences of spike-TfR interaction inhibited SARS-CoV-2 infection in Vero E6 cells and mouse model. Results Elevated expression of TfR in respiratory tract and lung tissue of monkey and mouse infected by SARS-CoV-2 TfR is ubiquitously expressed 23. Given that respiratory tract is a susceptible site for SARS-CoV-2 infection, we employed qRT-PCR and western blot techniques to detect the expression of TfR and ACE2 in several tissues including respiratory tract (nasal cavity, trachea, and lung) and liver. In both RNA and protein levels, the expression of 5 bioRxiv preprint doi: https://doi.org/10.1101/2020.10.23.350348; this version posted October 23, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. both TfR and ACE2 were significantly elevated in trachea and lung as compared with other tissues (Fig. 1A-C). As illustrated in Fig. 1D and E, TfR was upregulated in lung tissue of SARS-CoV-2 infected monkey and humanized ACE2 (hACE2) mice by immunohistochemical analysis. Direct interactions among virus spike protein, ACE2 and TfR Surface plasmon resonance (SPR) was used to study the interaction between TfR and the virus spike protein. As illustrated in Fig. 2A and B, TfR directly interacted with the virus spike protein through enzyme-linked immunosorbent assay (ELISA) and SPR analysis, whereby, the association rate constant (Ka), dissociation rate constant (Kd), and equilibrium dissociation constant (KD) values for the interaction between TfR and spike were 2.69× 105 M−1s−1, 7.92 × 10−4 s−1and 2.95 nM, respectively. The effect of TfR on the SARS-CoV-2 spike RBD, which binds to the cell receptor ACE224,25, was also assayed using SPR (Fig. 2C). The KD value between TfR and SARS-CoV-2 spike RBD was～43 nM, which is a bit weaker compared with the binding affinity between TfR and SARS-CoV-2 spike. Based on the TfR structures 26 and the virus spike protein 27, we made a docking model of TfR-spike interaction (Fig. S1). According to the model, two designed peptides (SL8: SKVEKLTL; QK8: QDSNWASK) were used to interfere with the binding of TfR to spike (Table S1). As illustrated in Fig. 2D, these peptides inhibited TfR-Spike interaction. As illustrated in Fig. 2E and F, TfR also directly interacted with ACE2 through 6 bioRxiv preprint doi: https://doi.org/10.1101/2020.10.23.350348; this version posted October 23, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. SPR and native polyacrylamidegel electrophoresis (PAGE) analysis, and the Ka, Kd, and KD values for the interaction between TfR and ACE2 were 6.33× 104 M−1s−1, 1.25 × 10−2 s−1 and 200 nM, respectively. Based on the TfR, ACE2, and spike protein structures, we made docking models of TfR-ACE2 (Fig. S2) and TfR-ACE2-Spike interactions (Fig. S3). According to these models, two inhibitory peptides (SL8 and FG8: FPFLAYSG) were designed to interfere with TfR-ACE2 complex formation (Table S2). As illustrated in Fig. 2G, these peptides inhibited TfR-ACE2 interaction determined by SPR.